Table 3.
Impact of metabolite instability on interpretation of amino acid concentrations.
| Effect of Individual AA on Retrospective Assessment | Assessment of Parameters/Disorders | Effect of AA Disease Ratios on Retrospective Assessment |
|||||
|---|---|---|---|---|---|---|---|
| Parameter * | Disorder | Retrospective Analysis of IMDs | RCVa | Annual Percentile Decay | RCVa Reached | Metabolite Ratio | Retrospective Analysis of IMDs |
| Risk Category | (%) | (%) | (years) | Risk Category | |||
| Arg | ARG | False-negative | 56.5 | - *** | - *** | Arg/Orn, Arg/Phe, Arg/Ala | Cannot be interpreted |
| Cit/Arg (low) | Cannot be interpreted | ||||||
| Cit (low) | OTC/CPS | False-positive | 31.9 | 8 | 4.0 | No informative AA disease ratios | - |
| NAGS, OAT | False-positive | 31.9 | 8 | 4.0 | No informative AA disease ratios | - | |
| Cit | CIT-I | False-negative | 31.9 | 8 | 4.0 | Cit/Phe | none |
| Cit/Arg, Met/Cit (low) | - | ||||||
| (Low): Ala/Cit, Glu/Cit, Orn/Cit | none | ||||||
| Pro/Cit (low) | False-negative | ||||||
| Gln/Cit (low) | False-positive | ||||||
| CIT-II | False-negative | 31.9 | 8 | 4.0 | Cit/Phe | none | |
| Pro/Cit (low) | False-negative | ||||||
| Gln/Cit (low) | False-positive | ||||||
| PC | False-negative | 31.9 | 8 | 4.0 | Cit/Phe | none | |
| Met/Cit (low) | - | ||||||
| (low): Ala/Cit, Glu/Cit, Orn/Cit | none | ||||||
| Pro/Cit (low) | False-negative | ||||||
| ASA | False-negative | 31.9 | 8 | 4.0 | (low): Cit/Phe, Ala/Cit | none | |
| Pro/Cit (low) | False-negative | ||||||
| Orn/Cit (low) | False-positive | ||||||
| Met/Cit (low) | - | ||||||
| Gln (low) | CIT-II | False-positive | 53.7 | Exponential decay (Table 2) | <1 | See CIT – CIT-II | |
| Glu (low) | PC | False-positive | 21.6 | 9 | 2.4 | See CIT-PC | |
| Gly (low) | 3PGDH | False-positive | 23.0 | 12 | 2.2 | Ala/(Ser + Gly) | False-positive |
| Gly | NKHG | False-negative | 23.0 | 12 | 2.2 | No informative AA disease ratios | |
| Xle ** | MSUD | False negative | 19.11 | 6 | 3.2 | Val/Phe, Xle/Phe, Xle/Ala, Xle/Tyr | False-positive |
| Met/Xle (low) | - | ||||||
| (Ile + Leu + Val)/(Phe + Tyr) | False-positive | ||||||
| Gly/(Ile + Leu + Val) | False-negative | ||||||
| Xle (low) | BCKDK | False-positive | 19.11 | 6 | 3.2 | (low): Val/Phe, Xle/Phe, Xle/Ala, (Xle + Val)/(Phe + Tyr) | False-negative |
| Met (low) | RMD | False-positive | 18.6 | - *** | - *** | Met/Phe (low) | Cannot be interpreted |
| Met | HCY | False negative | 18.6 | - *** | - *** | Met/Ala, Met/Xle, Met/Phe, Met/Cit, Met/Val, Met/Gly, Met/Pro | Cannot be interpreted |
| H-MET | 18.6 | - *** | - *** | Met/Ala, Met/Phe, Met/Cit, Met/Val, Met/Gly, Met/Tyr, Met/Pro, Met/Xle | Cannot be interpreted | ||
| Orn (low) | SSADH | False-positive | 55.1 | 10 | >5 | No informative AA disease ratios | |
| Phe | PKU, H-PHE, BIOPT (BS/Reg) | False-negative | 20.8 | 7 | 3.0 | Phe/Tyr, Cit/Phe (low) | none |
| (low): Val/Phe, Xle/Phe, (Ile + Leu + Val)/(Phe + Tyr) (PKU) | False-negative | ||||||
| (low): Met/Phe, Arg/Phe | - | ||||||
| Orn/Phe (low) | False-positive | ||||||
| Pro | H-PRO | False-negative | 15.8 | 4 | 4.0 | Pro/Cit | False-positive |
| Orn/Pro (low) | False-positive | ||||||
| Tyr | TTI, TTII, TTIII | False-negative | 15.8 | 6 | 2.6 | Tyr/Pro (TT2) | False-negative |
| Phe/Tyr, Xle/Tyr (low) | none | ||||||
| Met/Tyr (low) | - | ||||||
| (Ile + Leu + Val)/(Phe + Tyr) (low) | False-negative | ||||||
| Val | MSUD | False-negative | 19.11 | 5 | 3.8 | See Xle | |
| Val (low) | BCKDK | False-positive | 19.11 | 5 | 3.8 | See Xle (low) | |
* ‘Low’ is added if low values of the AA are indicative of the disease. If no indication is added, this indicates elevated values. ** Xle represents the combination of the isomers Leu, Ile and allo-Ile that are not separated using flow-injection MS/MS methods. *** Arg, His and Met concentrations were below LOQ in all samples in our study, rendering evaluation of ratios including these AA not feasible. Table 3 shows the consequences associated with the interpretation of retrospectively analyzed AA and AA ratios in DBS upon long-term storage at RT for the detection of IMDs, based on the CLIR-database. Abbreviations (in alphabetical order): ASA, arginino-succinic acidemia (#207900); ARG, argininemia (OMIM, #207800); BCKDK, Brached-Chain Keto Acid Dehydrogenase Kinas Deficiency (#614923); BIOPT (BS), disorders of biopterin biosynthesis (#261640); CB1, cobalamin (homocystinuria-megaloblastic anemia, complementation type (#236270) CIT-I, citrullinemia type I (#215700); CIT-II, citrullinemia type II (#605814, #603471); CPS, carbamyl-phosphate synthase deficiency (#237300); HCY, hyper-homo-cysteinemia (#603174); H-PHE, hyperphenylalaninemia (#261600); H-PRO, hyper-prolinemia (#239500); MSUD, maple syrup urine disease (#248600); H-MET, hyper-methioninemias (#250850); NAGS, N-acetyl-glutamate Synthase Deficiency (#237310); NKHG, nonketotic hyperglycinemia (#605899); OTC, ornithine trans-carbamylase deficiency (#300461); PKU, phenylketonuria (#261600); PC, pyruvate carboxylase deficiency (#266150); RMD, Remethylation defects (homocystinuria-megaloblastic anemia, CblG #250940 and CblE #236270 complementation types and (N)5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency #236250); SSADH, Succinic Semialdehyde Dehydrogenase Deficiency (#271980); TTI, tyrosinemia type I (#276700); TTIII, tyrosinemia type III (#276710); TTII, tyrosinemia type II (#276600); 3PGDH deficiency, Phosphoglycerate dehydrogenase deficiency (#601815). The AAs: Ala, Asn, Asp, His, Lys, Ser, Tau, Thr and Trp were not indicative disease markers and were therefore excluded from the table. The disorders: NKHG, TT1, NH, OAT, OTC/CPS and NAGS had no informative AA disease ratios. Assessment of Arg, Orn, and Gln was possibly less reliable due to high CVs (indicated in light grey).